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1. BOO E 1 E R S Seer Seer eae ReGen ai 0 0 e O U L 0 6 12 24 36 48 Treatment time hr u Fig 2 Concentrations of total human ISG15 in HeLa ce ate after FNa treatment for indicated times Time course of ISG15 production in HeLa cell lysate 160 Non treatment W400 Pt we Og ee ee Shee ee ee eee eee E IFNo treatment 120 SS Ss Se eee ee ee ee ee aa 100 ISG15 conc ng mL S 0 m f 0 2 4 6 8 12 24 48 Treatment time hr C CY 8085 15 Version 120710 Human ISG15 ELISA Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 IFNa dose dependent production of ISG15 in HeLa cell culture medium ISG15 level in HeLa cell culture medium after IFN a treatment 9 0 _ 8 0 F4 O24brtreatment 777777777777 7 0 f E 48 hr treatment E 0 0 A ea See Sh ee eae E E AM possercerccanesnas E E EOC 3 0 H E E E 20 H E E EMGO ISG15 conc ng mL 10 E E E E E EEO 0 0 0 0 0 3 1 6 8 0 40 0 200 0 1 000 0 5 000 0 IFNa conc U mL v Fig 4 IFNa dose dependent production of ISG15 T after IFNa treatment ISG15 level in HeLa2. All samples and standards should be assayed in te The following components are supplied and are sufficient for the one 96 well microplate kit Microplate One microplate supplied ready t ith 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated uman ISG15 antibody as a capture antibody 10X Wash Buffer One bottle containing 100 4L of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containi of 1X buffer use for and sample dilution Ready to use Human ISG15 Standard One yi aining 40 ng of lyophilized recombinant human ISG15 HRP conjugated Detectio conjugated anti human IS y One bottle containing 12 mL of HRP horseradish peroxidase dy Ready to use Substrate Reagent containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready t Stop Solution le containing 20 mL of 1 N H SOy Ready to use C CY 8085 3 Version 120710 Human ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e Vortex mixer Qy e Microplate washer optional Manual washing is possible but not preferable e Plate reader capable of measuring absorbance in 96 well plates a
3. Standard Assay Procedure for Human ISG15 j Remove the appropriate number of microtiter wells from the foil pouch and place them into the holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Dilute samples with Dilution Buffer See Sample Preparation above Pipette 100 uL of Standard Solutions Std1 Std7 Blank and the diluted samples tes into the appropriate wells Incubate the wells at room temperature ca 25 C for 2 hours shaking at ca 3 on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash Buffer 350 uL using a s le multi channel pipette manifold dispenser or microplate washer w D 6 Add 100 uL of HRP conjugated Detection Antibody into each well N Incubate the wells at room temperature ca 25 C for 2 hours at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each well with Wash Buffer 350 u sing squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent to each sunlight Covering the wells with e g aluminu 4 C immediately after the necessary volume is re AV posing the microtiter plate to direct recommended Return Substrate Reagent to 10 Incubate the wells at room temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation ay be extended up to 30 minutes if the reaction
4. temperature is below than 20 C 11 Add 100 uL of Stop Solution to each w ll in the same order as the previously added Substrate Reagent 12 Measure absorbance in each we ing a spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelen 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wa an be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete remoyal of liquid at each step is essential to good performance After the last wash Note 2 Reliabl ard curves are obtained when either O D values do not exceed 0 25 units for the oncentration or 3 0 units for the highest standard concentration The plate nitored at 5 minute intervals for approximately 30 minutes plate reader is not capable of reading absorbance greater than the absorbance of the andard perform a second reading at 405 nm A new standard curve constructed e values measured at 405 nm is used to determine ISG15 concentration of off scale amples The readings at 405 nm should not replace the on scale readings at 450 nm CY 8085 120710 Version Human ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Calculations Average the duplicate readings for each Standard Solution control and sample and subtrac average zero standard optical density Plot the optical density for the stan
5. ISG15 Cat CY 72 PRODUCED BY cA CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 761 CycLex Circ ucts are supplied for research use only CycLex CircuLex products and components may not be resold modified for resale or used to manufacture commercial ior written approval from CycLex Co Ltd To inquire about licensing for al use please contact us via email CY 8085 Version 120710
6. Upon receipt store all ca e Don t expose reagents C CY 8085 1 Version 120710 Human ISG15 ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction ISG15 Interferon IFN Stimulated Gene 15 is a ubiquitin like protein containing two ubi homology domains and becomes conjugated to a variety of proteins when cells are treated wit interferon or lipopolysaccharide Therefore this modification so called SGylation involved in type I interferon signal transduction Unlike ubiquitylation SGylation does no degradation regulated by K48 linked ubiquitin ISG15 is expressed as a 165 amino acid is subsequently processed to expose the C terminal sequence LRLRGG Like ubiquitin diglycine residue is adenlylated and conjugated by a thiolester bond to sequential cystine residt El activating E2 carrier and E3 ligase enzymes before being transferred to lysine gesidues on target protein substrates ISGI5 can also be found extracellularly in an unconjugated form free form an lymphocytes and monocytes were reported to release free ISG15 following treatment with than 50 of the total ISG15 could be detected in the culture medium after 24 hours followa stimulation In addition ISG15 is highly elevated and extensively conjugated to cellular ems many tumors and tumor cell lines The increased levels of ISG15 in tumor cells were to be associated with decreased levels of polyubi
7. cell lysate after IFNa treatment 140 120 O24 hr treatment 3 E 100 E48 hr treatment i Sb 80 Q 5 60 2 pi A 40 20 0 0 0 1 6 8 0 40 0 200 0 1 000 0 IFNa conc U mL C CY 8085 16 Version 120710 Human ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References Korant BD Blomstrom DC Jonak GJ Knight E Jr 1984 J Biol Chem 259 14835 14839 2 Blomstrom DC Fahey D Kutny R Korant BD Knight E Jr 1986 J Biol Chem 261 881 w Haas AL Ahrens P Bright PM Ankel H 1987 J Biol Chem 1987 262 11315 11323 4 Feltham N Hillman M Jr Cordova B Fahey D Larsen B Blomstrom DC amp K r E Jr 1989 J Interferon Res 9 493 507 Knight E Jr amp B Cordova 1991 J Immunol 146 2280 2284 Qy 6 Loeb K L amp Haas A L 1992 J Biol Chem 267 7806 7813 Nn 7 D Cunha J Knight E Jr Haas A L Truitt R L amp Borden EC 1996 atl Acad Sci USA 93 211 215 8 D Cunha J Ramanujam S Wagner R J Witt PL Knight E rden EC 1996 J Immunol 157 4100 4108 9 Austin K J S K Ward M G Teixeira Dean VC Moo 54 600 606 ansen TR 1996 Biol Reprod 10 Kelso A 1998 Immunol Cell Biol 76 300 317 11 Durfee Larissa A Lyon Nancy Seo Kyungw and Huibregtse Jon M 2010 Mol Cell 38 722 732 Related Products CircuLex Mouse ISG15 ELISA Kit 091 CircuLex Human
8. Aliquots of plasma may als red at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Note Citrate plasma has not been validated for use in this assay Other biological samples Remove any particulates by centrifugation a ess immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles C CY 8085 6 Version 120710 Human ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Human ISG15 ELISA Kit is provided with remo strips of wells so the assay can be carried out on separate occasions using only the number o required for the particular determination Since experimental conditions may vary an algue human ISG15 Standard within the kit should be included in each assay as a calibratg pipette tips and reagent troughs should be used for all liquid transfers to avoid cross co reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r are supplied ready to use with the exception of 10X Wash Buffer and Human ISG15 Stand 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X fer to 900 mL of deionized distilled water ddH O Mix well Store at 4 C for two off 20 C for long term storage 2 Reconstitute Human ISG15 Standard with 1 0 mL of ddH O ISG15 in vial sho
9. Human ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human ISG15 CircuLex Human ISG15 ELISA Kit cA Cat CY 8085 O Intended Use cisssiesadstatatcssassaaaiaaabalenatoiatons 1 EASE EEEE 1 TREOOU CUI OM a iesscascsnceadatacatantonatabotectacincevedeads 2 Principle of the Assay 2 3 Materials Provided pissiccsecsssanconcccsasndecancesuaneds 3 Materials Required but not Provided 4 Precautions and Recommendation 5 QO Sample Collection and Storage 0 6 Detailed Proto ls sassccncsseseaverreascastiamareconootes 7 8 Calculations cass cocarcersecassucesqunaudeneedinvedeaseaaaey 9 Measurement Range eceeeeeeseseeeeeeeeeeee 9 TrOubleshOOtii s icccissasssssneccenavasonanssassesanaeen 9 Reagent SCADUILY vancarsnasasacesansaanmecasesadonanagis 9 Sample PreparattO ccsevesccssecsssasssecsecvonessnes 10 Assay Characteristics cccssssscccscacsscssesecessesees Example of Test Results eee eeeeeeereeee 1 IRGICTCHICES s senanstaneecaanedscbecvensennnenseenstaccadawns 17 Related PROGUCIS viisisscovestteacvsaiennsnmesnveang 7 Intended Use The CycLex Research Product Circulex Human ISG15 ELISA Kit is used for the quantitative measurement of human ISG15 in cell lysate culture medium and human serum This assay kit is for research use nly and not for use in diagnostic or therapeutic procedures Storage e
10. Human ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Inter assay Precision Precision between assays Three samples of known concentration were tested in five separate assays to assess inter ass precision e Inter assay Run to Run n 5 CV 2 9 7 2 Human ISG15 concentration ng mL O Sample 1 Sample 2 Sample 3 1 5 80 3 02 0 90 2 5 53 2 87 0 83 Qy 3 5 11 2 90 0 79 4 5 09 2 90 0 5 5 24 3 06 3 Linearity To assess the linearity of the assay samples co g r spiked with high concentrations of unconjugated free form human ISGI5 were s iluted with the Dilution Buffer to produce samples with values within the dynamic range of th Linearity Ag peeetpiebe ieee a e Sample 1 A Sample 2 2 3 0 Sample 3 d 5 o c ro 2 0 9 oO 2 c E10 0 0 0 0 2 0 4 0 6 0 8 1 1 2 Sample Dilution Ratio C CY 8085 14 Version 120710 Human ISG15 ELISA Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Concentrations of unconjugated free form human ISG15 in HeLa cell culture medium after treatment for indicated times Time course of ISG15 production in HeLa cell mediun 6 0 Non tre atme nt 3 5 0 IFNa treatment OStstsSt CS lt CS CSsS E AO E E ee aa eee eee anaeeuaauaing g SSO ee ee R N
11. cell lines and is provided as an example of sufi methods It is strongly advised that the user always perform an initial experiment to determinesthe dilution to be used in subsequent experiments This need not be any more than a single tin using serial dilutions of the cell lysate One eight well strip of the substrate plate should b this initial experiment All steps of cell lysate preparation should be performed at 4 C and lysates should be kept at below 70 C Preparation of Cell Lysate Qy A Preparation of Cell Lysis Buffer 20 mM Tris HCI pH 7 5 250 mM NaCl 10 glycerol 0 1 NP 40 1 A 1 mM EGTA 0 2 mM PMSF 1 ug mL pepstatin 0 5 ug mL leupeptin 0 2 mM DTT B Preparation of poly l lysine coated plate Coat the plate with 25 ug mL poly l lysine PLL in PBS for 4 C Subsequently go to a washing step with PBS C Treatment of Cells 1 Plate adherent cells in PLL coated 96 well plate at around 1 ells well 2 Incubate the culture dish at 37 C for 7 12 hours i c r 3 Add appropriate amount of test compound or IFN vehicle for test compound to each well 4 Incubate the culture dish at 37 C for appropriate time D Cell Extraction Note This protocol has been successfully applied to HeLa cell line Users should optimize the cell extraction procedure for thei cations 1 Wash cells three times with Ae Remove any remaining PBS by decanting Invert the plate and blot it against clean pa At this point the cells in the plate ca
12. dards versus the conce io of the standards and draw the best curve The data can be linearized by using log log d regression analysis may be applied to the log transformation To determine the concentration of each sample first find the absorbance value on the y axis and extend a line to the standard curve At the point of intersection extend a vertical line to the x axis the corresponding human ISG15 concentration If the samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor 1 The dose response curve of this assay fits best to a sigmoidal four parameter 1 results of unknown samples can be calculated with any computer program logistic function It is important to make an appropriate mathematical adjus the dilution factor our parameter accommodate for 2 Most microtiter plate readers perform automatic calculations of analyte entration The calibration curve is constructed by plotting the absorbance Y of calib sus log of the known concentration X of calibrators using the four parameter fi tion Alternatively the logit log function can be used to linearize the calibration curve i e logit efyabsorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 0 188 ng mL to 12 standard should be diluted with Dilution Buffer in to be taken into consideration in calculating the hum ample read
13. idic Stop Solution and Subs Solution which contains hydrogen peroxide e Wear gloves and eye protection when han unodiagnostic materials and samples of human origin and these reagents In case of conta th the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek m l attention when necessary Biological samples may be cont ith infectious agents Do not ingest expose to open wounds or breathe aerosols W ective gloves and dispose of biological samples properly handling Sto Q e CAUTION Sulfuric See g acid Wear disposable gloves and eye protection when n C CY 8085 5 Version 120710 Human ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Serum Use a serum separator tube and allow samples to clot for 60 30 minutes Centrifug samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or store sam 0 ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 C e periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Nay as the anticoagulant If possible collect the toa mixture of EDTA Na and Futhan5 to stabilize the sample against spontaneous in vitro complement activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x g Assayeimmediately or store samples on ice for up to 6 hours before assaying
14. ing higher than the highest dilution and re assayed Dilution factors need G15 concentration Troubleshooting 1 The Standard Solutions should be m cate using the protocol described in the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 2 Poor duplicates accompanied by elev values for wells containing no sample indicate insufficient washing If all instructions i iled Protocol were followed accurately such results indicate a need for washer maintenan 3 Overall low signal may ndic at desiccation of the plate has occurred between the final wash and addition of Substrate Re after wash t Do not allow the plate to dry out Add Substrate Reagent immediately included in the CycLex Research Product CircuLex Human ISG15 ELISA Kit have b stability Reagents should not be used beyond the stated expiration date Upon recel agents should be stored at 4 C except the reconstituted ISG15 Standard must be stored at belo e Microplate should be stored in the original foil bag sealed by the zip lock and contain desiccant pack C CY 8085 9 Version 120710 Human ISG15 ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation Several extraction methods can be used for measurement cellular total ISG15 The following pro has been shown to work with a number of different
15. n be frozen at below 70 C and lysed at a later date 2 Lyse the cells by addin 300 rpm by an orbitalgmi 1 mL of Cell Lysis Buffer for 60 90 minutes at 4 C with rotating at ca te shaker To get a ro could adjust the cell concentration to around 1 2 x 10 cells mL in Cell Lysis AR protein concentration of the HeLa cell lysate should be 0 03 0 04 mg mL using this ropriate volume of Cell Lysis Buffer depends on the cell line the cell number and the total ISG15 For example 1 2 x 10 HeLa cells can be lysed in 0 1 mL of Cell Lysis 120710 CY 8085 Version Human ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 3 Centrifuge at 3 500 rpm for 15 minutes at 4 C using a microplate bucket or transfer the cell lys to microcentrifuge tubes and centrifuge at 15 000 rpm for 5 minutes at 4 C 4 Transfer the clear cell lysates to new 96 well microplate or clean microcentrifuge tubes Dilute the cell lysates 10 times with Dilution Buffer 100 uL of these diluted cell lysates are ready forass o to the section Standard Assay Procedure for Human ISG15 at page 8 Typical data using this protocol are shown in Fig 2 and Fig 4 page 15 and 16 The cell lysates can be stored at below 70 C Avoid multiple freeze thaw cycles After thaw the cell lysates Centrifuge at 15 000 rpm for 5 minutes at 4 C again since the cell lysates s e clear of an
16. quitinated proteins Principle of the Assay The CycLex Research Product CircuLex Human ISG1 sandwich enzyme immunoassay technique An antibody spe microplate Standards and samples are pipetted into the wells human ISG15 present After washing away any u it employs the quantitative uman ISG15 is pre coated onto a the immobilized antibody binds any nces an HRP conjugated antibody to remove any unbound antibody HRP ith the substrate H O tetramethylbenzidine specific for human ISG15 is added to the wells Fo conjugate the remaining conjugate is allowed to The reaction is stopped by addition of acidic solutio absorbance of the resulting yellow product is measured at 450 nm The absorbance is proportional to concentration of human ISG15 A standard curve is constructed by plotting absorbance vy versus human ISG15 concentrations of calibrators and concentrations of unknown samples are di d using this standard curve C CY 8085 2 Version 120710 Human ISG15 ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted samples to the wells Yy Incubate for 2 hours at room temp cA Wash the wells O v Add 100 uL of HRP conjugated anti human ISG15 antibody v Incubate for 2 hours at room temp Wash the wells Add 100 uL of Substrate Reagent y Add 100 uL of Stop Solution v Measure absorbance at 450 nm Materials Provided
17. t dual e s of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used The pl so be read at a single wavelength of 450 nm which will give a somewhat higher reading e Software package facilitating data generation and analysis opti 500 or 1000 mL graduated cylinder e Reagent reservoirs e Deionized water of the highest quality e Disposable paper towels C CY 8085 4 Version 120710 Human ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Allow all the components to come to room temperature before use e All microplate strips that are not immediately required should be returned to the zip lock h must be carefully resealed to avoid moisture absorption e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit e Rinse all detergent residues from glassware Qy e Use deionized water of the highest quality e Do not mix reagents from different kits The buffers and reagents used in this kit contain NaN as preservati hould be taken to avoid direct contact with these reagents Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the N where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containi ions in compliance with local regulations e Avoid contact with the ac
18. uld be 40 ng mL which is referred as a Master centration of the human d of human ISG15 Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series the next transfer The 12 ng mL standard Std 1 serve Buffer serves as the zero standard Blank OW Mix each tube thoroughly before the highest standard The Dilution Volume of Standard ilution Buffer Concentration Std 1 180 uL of Master Standard 420 uL 12 0 ng mL Std 2 300 uL of Std 1 12 0 ng 300 uL 6 0 ng mL Std 3 300 uL of Std 2 6 0 ng 300 uL 3 0ng mL Std 4 300 uL of Std 3 3 0 300 uL 1 5 ng mL Std 5 300 uL of Std 4 1 300 uL 0 75 ng mL Std 6 300 uL of Std 5407 300 uL 0 375 ng mL Std 7 300 uL of Std46 0 ng mL 300 uL 0 188 ng mL Blank 300 uL 0 ng mL Note Do not use a Rep ipette Change tips for every dilution Wet tip with Dilution Buffer before dispensing Unused portions of Master Standard should be aliquoted and stored at below 70 C immediately id multiple freeze and thaw cycles Sample Preparati e Optimal diluti 1 conditioned medium for measurement of ISG15 depends on cell number and IFN does eat to appropriate dilution plasma samples require a 10 40 fold dilution depending on ISG15 concentration in sample CY 8085 Version 120710 Human ISG15 ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures
19. y sediments or particulate matter NOTE THE ABOVE PROCEDURES ARE INTENDED ONLY AS haps THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY ING ON THE PARAMETERS BEING INVESTIGATED AND MUST B INED BY THE INDIVIDUAL USER NO WARRANTY OR GUARAN PERFORMANCE USING THESE PROCEDURES IS MADE OR ae C CY 8085 11 Version 120710 Human ISG15 ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of human ISG15 giving absorbance highes tha mean absorbance of blank plus three standard deviations of the absorbance of blank A blank is better than 0 85 ng ml of sample Dilution Buffer is pipetted into blank wells Typical Standard Curve Q Human IGS15 Standard Curve s To N A S E E E ied oa oa 75 10 12 5 Human ISG15 conc ng ml C CY 8085 12 Version 120710 Human ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested sixteen times on one plate to assess intrazass precision e Intra assay Within Run n 16 CV 1 3 4 6 O Human ISG15 concentration ng mL Sampel Sample 2 Sample 3 TBSL DUAR WN N ee DuA Uw C CY 8085 13 Version 120710
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